ORIGINAL RESEARCH ARTICLE
Intervertebral Disc Degeneration in a Percutaneous Mouse Tail Injury Model Zuozhen Tian, MS, Xiaoyuan Ma, MD, Miersalijiang Yasen, MD, PhD, Robert L. Mauck, PhD, Ling Qin, PhD, Frances S. Shofer, PhD, Lachlan J. Smith, PhD, Maurizio Pacifici, PhD, Motomi Enomoto-Iwamoto, DDS, PhD, and Yejia Zhang, MD, PhD Objectives: Intervertebral disc (IVD) degenerates progressively with age and after injuries. In this study, we aimed to characterize early molecular events underlying disc degeneration using a mouse tail IVD injury model. Design: We have established a transcutaneous minimally invasive approach to induce mouse tail IVD injury under fluoroscopic guidance. Morphological and molecular changes in the injured IVDs are compared with the baseline features of adjacent intact levels. Results: After needle puncture, tail IVDs exhibited time-dependent histological changes. The aggrecan neoepitope VDIPEN was evident from 2 days to 4 wks after injury. A disintegrin and metalloproteinase domain-containing protein 8 (adam8) is a surface protease known to cleave fibronectin in the IVD. Gene expression of adam8 was elevated at all time points after injury, whereas the increase of C-X-C motif chemokine ligand (cxcl)-1 gene expression was statistically significant at 2 days and 2 wks after injury. Type 1 collagen gene expression decreased initially at day 2 but increased at 2 wks after injury, whereas no significant change in type 2 collagen gene expression was observed. The extracellular matrix gene expression pattern is consistent with fibrocartilage formation after injury. Conclusions: Mouse tail IVDs degenerate after needle puncture, as demonstrated by histological changes and aggrecan degradation. The minimally invasive tail IVD injury model should prove useful to investigators studying mechanisms of IVD degeneration and repair. Key Words: Intervertebral Disc, Mouse Model, Injury, Degeneration, A Disintegrin and Metalloproteinase Domain-Containing Protein 8 (Am J Phys Med Rehabil 2017;00:00–00)
n humans, because most patients do not receive medical attention for back pain during the early stages of intervertebral disc (IVD) degeneration, little is known about the changes that occur during the initial phases of such degeneration. Animal models of disc degeneration would be useful in understanding the events that initiate inflammation/repair and in identifying critical steps that may be amenable to intervention.1 This work aims to define the early molecular changes during the initial 4 wks, concurrent with histological changes after an injury to the tail IVD in the mouse. The IVD injury model has been widely used to study the course of IVD degeneration, in large animals (ovine,2 porcine3),
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dogs,4 and small animals (rabbit,5–8 rat,9 and mouse10,11). One main advantage of the mouse model is the ability to perform genetic manipulations during IVD degeneration. Challenges of working with the mouse IVD mainly reflect its small size: surgical precision is crucial, and the amount of tissue for molecular and biochemical assays is limited. The IVD histology and molecular changes after injury with a 29-gauge (G) needle were first described by Yang et al.11 The deterioration of biomechanical properties along with histological changes after tail IVD injury with a 26-G needle were further described by Martin et al.10 The needle puncture, with a 26-G needle, was severe enough to induce IVD degeneration detectable with biomechanical
From the Department of Physical Medicine & Rehabilitation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (ZT, MY, RLM, YZ); Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (XM, RLM, LQ, LJS, YZ); Translational Musculoskeletal Research Center, Corporal Michael J. Crescenz Veterans Affairs Medical Center, Philadelphia, Pennsylvania (RLM, LJS, YZ); Department of Emergency Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (FSS); Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (LJS); Department of Surgery, Division of Orthopaedic Surgery, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania (MP, ME-I); and Department of Orthopaedics, University of Maryland, Baltimore, Maryland (ME-I). All correspondence and requests for reprints should be addressed to: Yejia Zhang, MD, PhD, Department of Physical Medicine & Rehabilitation, Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, and Translational Musculoskeletal Research Center, Corporal Michael J. Crescenz Veterans Affairs Medical Center, Philadelphia, PA 19104. YZ has been supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (1K08 HD049598). This work is supported, in part, by research grants from the North American Spine Society, a pilot grant
from the Department of Veterans Affairs Healthcare Network-VISN 4, and a Penn Center for Musculoskeletal Disorders pilot grant (P30-AR050950-10 Pilot). All authors critically reviewed and approved the article. The Thomas Jefferson University Sidney Kimmel Cancer Center Translational Research/Pathology Shared Resource Core Facility provided outstanding histology service. None of the authors have any professional and financial affiliation that could be perceived to be biased in the presentation of the current work. ZT, MY, XM, LQ, ME-I, and YZ contributed in data acquisition. ZT, YZ, MP, ME-I, LJS, and RLM contributed in research design and/or data interpretation. FSS contributed in statistical analysis and research design. YZ, MP, ME-I, LJS, RLM, and LQ drafted and revised the article. YZ and ZT are responsible for storage and access of all primary data. All authors have read and approved the final submitted article. Financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.ajpmr.com). Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0894-9115 DOI: 10.1097/PHM.0000000000000818
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measures.10 The advantages of the tail injury method include easy access to the tail disc, low morbidity, and a reproducible course of degeneration.
Primary Objective The main objective of this study was to further refine the surgical technique, including percutaneous puncture of the disc, thus shortening and simplifying the procedure and reducing mouse suffering. As a result of reduced surgical trauma, confounding factors are also reduced in the examination of inflammatory markers. Using this IVD injury model, we determined the time course of histopathological changes and changes in gene expression of adam8, which is an important molecule in inflammation12,13 and is upregulated in degenerated IVDs in humans.14 We also examined cxcl-1 gene expression after injury because we have previously shown that interleukin 8, the human homologue of cxcl-1, is elevated in human IVD tissues surgically removed because of back pain.15
MATERIALS AND METHODS Mice All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Corporal Michael J. Crescenz Veterans Affairs Medical Center in Philadelphia (approval # 01517-0002). Fifteen young adult (8 wk old) female C57BL/6j mice (the Jackson Laboratory, Bar Harbor, ME) were used in this study. Mice were housed under pathogen-free conditions with environmental enrichment, with up to five animals per cage. Twenty animals were used for RNA extraction and histology, with five animals at each of four time points.
Tail Injury Surgery The mouse was anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg) subcutaneously. Under anesthesia, the mouse tail IVDs were injured with a 26-G needle inserted under fluoroscopic guidance with a mini C-arm (OrthoScan FD Pulse Mini C-Arm, Orthoscan Inc, Scottsdale, AZ). Specifically, the skin was cleaned with betadine, the mouse coccygeal
(Co) IVDs were identified, and a 26-G needle was inserted into the IVD space until the needle tip reached 2/3 of the disc thickness (Fig. S1, Supplemental Digital Content 1, http://links. lww.com/PHM/A472). Gelatinous tissues were often found on the needle tip after this had been removed, suggesting that the needle puncture induces an acute herniation of the gelatinous nucleus pulposus (NP) of the IVD. An arrow indicates the coccygeal disc between the third and fourth coccygeal vertebrae (Co3/4; arrows in Fig. S1, Supplemental Digital Content 1, http://links.lww.com/PHM/A472). This procedure has been modified from previously published work,10 where a skin incision was made in the tail over the IVD before needle insertion into the disc. In the current study, the Co3/4 and Co5/6 IVDs in each mouse were injured, whereas Co4/5 and Co6/7 served as intact controls (Fig. S1, Supplemental Digital Content 1, http://links.lww.com/PHM/A472). The percutaneous approach reduced procedure time and minimized risk to the animals. The animals behaved normally (by observing breathing, eating, ambulation, etc.) and did not require medication for pain on the day after surgery. No adverse event such as infection or bleeding was noted. Five animals were sacrificed by exposure to CO2 at each of the following time points: 2 days, 1 wk, 2 wks, and 4 wks after tail disc injury. From each mouse tail, Co3/4 (injured) and Co4/5 (intact control) discs were isolated individually for RNA extraction. Co5/6 (injured) and Co6/7 (intact control) were isolated en bloc (Fig. 1) for histological examination and immunostaining (Fig. 3).
Histological Evaluation The IVDs and portions of the adjacent bony vertebral bodies were isolated immediately after euthanasia. The disc with its surrounding vertebral bodies was fixed with 4% paraformaldehyde for 24 hrs. The bone-disc-bone segments were decalcified with a solution consisting of 10% citric acid with 22% formic acid for 24 hrs until the bony portion was completely decalcified, as previously described.16 The tissues were then dehydrated and embedded in paraffin and sectioned to 5-μM thickness, and the sections were stained with Alcian blue with hematoxylin and eosin (H&E) counter stain or with H&E only. Specifically, the tissue sections were stained with
FIGURE 1. Histological changes (low magnification) 2 days, 1, 2, and 4 wks after coccygeal IVD injury. Injured (outlined in red) and the adjacent intact (outlined in green) IVDs were stained with Alcian blue and counterstained with H&E. Scale bars represent 1 mm.
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1% Alcian blue solution (Poly Scientific R&D Corp., Bay Shore, NY) for 5 minutes, followed by hematoxylin for 5 mins and eosin for approximately 20 secs. All samples were examined under a light microscope (Nikon) and photographed. Representative sections from each time point were chosen for presentation.
Histological Grades A modified histological grading scale for mouse discs was developed based on the previous grading methods.11,17 As shown in Table 1, a numeric scale for quantification of the degree of degeneration was designed. Analysis included the status of the annulus fibrosus (AF) organization and the extracellular matrix and cell morphology in the NP. Total scores ranged from 0 to 8. Scores of 0 to 2.0 represent essentially normal tissue; IVDs with mild disc degeneration scored 2.1 to 4.0 points; IVDs with moderate degeneration scored 4.1 to 6.0; IVDs with severe degeneration scored 6.1 to 8.0. Sections of injured and intact discs from 12 mice were evaluated by three experienced clinician scientists in a double-blinded manner. The total scores for NP and AF were calculated, and interobserver variability was assessed by a weighted Kappa using SAS software (Version 9.4; SAS Institute, Cary, NC). Average scores of all three raters were used to compare injured and intact disc histological grades.
Immunohistochemical Staining Aggrecan neoepitope was examined with antibody to its cleavage site, with the VDIPEN neoepitope at the cleavage site. Specifically, the paraffin sections were pretreated with 0.1% pepsin with 0.02 N HCl and incubated with primary antibodies (a generous gift from Dr. J. Mort, Shriner's Hospital) at 1:1000 dilution. The antibodies were visualized by incubation with appropriate biotinylated antibodies (Vector Laboratories, Burlingame, CA) followed by the color development method using the ImmpPCAT DAB Peroxidase Substrate kit (Vector Laboratories, Burlingame, CA). The sections were counterstained with hematoxylin.
RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction Intervertebral disc tissues were separated from their adjacent cartilaginous endplates and bone with a scalpel, under a
TABLE 1. Histological scoring criteria Grade 0 1 2 3 4
AF
NP
Discrete and well-apposed lamellae Moderately serpentine with rupture Severely serpentine with rupture, reversed contour Indistinct border of NP and AF Clefts formation; lamella fragmentation
Oval shape, highly hydrated, many notochordal cells Irregular shape, decreased hydration Large defects, cell clusters present Minor clefts, cell clusters decrease Occupied by connective tissue
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dissecting microscope (VistaVision; VWR International, Radnor, PA) (Fig. S2, Supplemental Digital Content 2, http://links.lww. com/PHM/A473). Total cellular RNA was isolated by the Trizol method as described previously.8 Specifically, the isolated IVD tissues were soaked in RNALater (Ambion, Foster City, CA) overnight, and stored at −80°C until extraction. On the day of RNA extraction, RNALater was removed and the tissues were snap frozen with liquid nitrogen, then transferred into Trizol (Invitrogen, Carlsbad, CA). The tissues were homogenized with a homogenizer, with disposable OmniTip probes for hard tissue (Omin International, Kennesaw, GA). Phenol was then extracted with chloroform three times and discarded. RNA was precipitated with 70% ethanol and was further purified using an RNeasy Micro Kit (Qiagen), according to the manufacturer's protocol. RNA concentration was determined using a Synergy H4 Hybrid Reader (BioTek, Winooski, VT). To generate cDNA, all RNA from each IVD (7–20 ng/μl, total volume 50 μl per sample) was used as template in a reverse transcriptase reaction using the SuperScript VILO cDNA synthesis kit (Life Technologies, Carlsbad, CA) containing random hexamers and added polyDT primers (Invitrogen, Carlsbad, CA). CDNA sequences were retrieved from Ensembl (release 84, March 2016). Primers for real-time polymerase chain reaction (PCR) (Table 2) were designed using Primer-BLAST18 and synthesized by Invitrogen (Carlsbad, CA) (Table 2). For each PCR reaction, cDNA, SYBR Select master mix (Life Technologies, Carlsbad, CA), and primers (working concentration 0.5 μM) were mixed, and deionized water was added to a total volume of 20 μl per reaction. MicroAmp Optical 96-well reaction plates (Applied Biosystems, Foster City, CA) with 20 μl of reaction mix/well were sealed with optical adhesive film (Life Technologies, Frederick, MD) and run in a ViiA7 real-time PCR system (Applied Biosystems, Foster City, CA) using the following program: (1) 50°C for 2 mins, (2) 95°C for 2 mins, (3) 95°C for 15 secs, (4) 58°C for 1 min, (5) repeat steps 3 and 4 for a total of 40 times. Single products were confirmed by determining melting curves at the conclusion of the reaction. Relative expression was calculated using the 2−ΔΔCt method19,20 normalized to gapdh (endogenous loading control).
Statistics The difference in genes of interest (adam8, cxcl1, col1a1, and col2a1) and house-keeping gene (gapdh) cycle thresholds (ΔCT) was calculated for each injured/intact pair. To assess differences in ΔCT between injured and intact tissues for each time period, a two-factor analysis of variance in repeated measures was used, where time was a grouping factor and injured/ intact was the repeated measure. Post hoc t tests using the pooled variance were performed for injured/intact differences within each time period. A P value of less than 0.05 was considered statistically significant. All analyses were performed using SAS statistical software (Version 9.4; SAS Institute, Cary, NC).
RESULTS Histology of Injured Mouse Tail IVD After injury, the IVD tissue showed loss of normal NP architecture (Figs. 1, 2). During the initial postinjury period www.ajpmr.com
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TABLE 2. Primers for real-time PCR Gene
Sequence
Expected Product
Transcript ID
Gapdh
F: 5′-AAG GGC TCA TGA CCA CAG TC-3′ R: 5′-GGA TGA CCT TGC CCA CAG-3′ F: 5′-TTG GCA CAG GCA GCA ACA TT-3′ R: 5′-CAC TCC CTC TTG TGG TTG CA-3′ F: 5′-CTG ACG CAT GGC CAA GAA GA-3′ R: 5′-ATA CCT CGG GTT TCC ACG TC-3′ F: 5′-CTG GGA ATG TCC TCT GCG ATG-3′ R: 5′-GCC CCT TTG GCC CTA ATT TTC-3′ F: 5′-TGCACCCAAACCGAAGTCAT-3′ R: 5′-TTGTCAGAAGCCAGCGTTCAC-3′
133 bp
ENSMUST00000118875 NM_008084 ENSMUST00000106069 NM_007403 ENSMUST00000001547 NM_007742 ENSMUST00000023123 NM_031163 ENSMUST00000031327 NM_008176
Adam8 Col1a1 Col2a1 Cxcl1
168 bp 72 bp 135 bp 177 bp
ENSMUST, Ensembl ID; F, forward; NM, The National Center for Biotechnology Information reference sequence; R, reverse.
(2 days to 2 wks), the empty spaces in the NP are most likely due to herniated NP, although this could also be the result of sectioning artifact (Fig. 2, red squares in 2-day, 1-wk and 2-wk panels and Figs. 2A–C and A′–C′, arrows). The whole IVD changed from an oval shape (Figs. 2E–H) to an irregular shape (Figs. 2A–D), suggesting loss of NP turgor, as a result of decompression. By 4 wks after injury, the IVD space was filled with chondrocyte-like cells (Figs. 2D, D′) positive to Alcian blue staining. The AF structure also changed from well-organized lamellae (Figs. 2E–H, yellow squares E″–H″) to severely serpentine layers with clefts (Figs. 2A–D, yellow squares and A″–D″). We evaluated histologic grade of the injured and control IVDs, modified from the grading system reported previously (Table 1).11 The scoring is based on microscopic sections, stained with Alcian blue and H&E (Fig. 2), and with H&E only (images not shown). Nucleus pulposus or AF showed grades ranging from 0 (normal) to 4 (severely degenerative), with the total score (NP + AF scores) ranging from 0 to 8 (Table 1). The scores of 3 graders correlated well, as measured by interrater variability (κ = 0.9311). The average scores for the intact IVDs ranged from 0.33 to 0.56, whereas those for the injured IVDs increased from 2.56 to 5.78 over time after the surgery (Table 3). The scores for intact discs were all in the normal range (≤2.0), whereas the scores for injured discs were in the mild (2.1–4.0) or moderate (4.1–6.0) range of degeneration according to the grading system described (Table 1). Differences between the histological scores of injured IVDs at 2 days, 1 wk, and 2 wks after injury, compared with those of intact IVDs, were not statistically significant (n = 3, P = 0.102, 0.099, and 0.077, respectively) (Table 3). The difference in histological score between injured and intact control IVDs was statistically significant at 4 wks after injury (n = 3, P = 0.0241) (Table 3).
Increase in Cleaved Products of Aggrecan Associated With Cartilaginous Changes Immunohistochemical examination was performed with an antibody that recognizes the aggrecan neoepitope, likely generated by matrix metalloproteinases (MMPs).21–23 Immunoreactivity was found in the regions rich in chondrocyte-like
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cells in injured IVD at 1 and 4 wks after injury (Figs. 3A, B, A′, B′; blue arrows), whereas the staining was limited to the interface between the NP and AF in intact IVDs (Fig. 3, intact′; blue arrows), suggesting that proteoglycan remodeling becomes active in the cartilaginous lesion in injured IVD.
Adam8 Gene Expression Is Upregulated After Injury We have previously demonstrated that disc degeneration is associated with increased adam8 protease and its fibronectin cleavage products in humans.14 To determine whether this phenomenon is recapitulated in this IVD injury model, adam8 expression was measured via quantitative real-time PCR for a 4-wk period after injury (Fig. 4A). After injury, adam8 gene expression increased compared with adjacent intact controls at all time points examined: day 2, P = 0.004; 1 wk, P = 0.018; 2 wks, P = 0.003; and 4 wks, P = 0.025 (n = 5 at each time point) (Fig. 4A).
Cxcl-1 Gene Expression Is Upregulated After Injury We have previously shown that interleukin 8, the human homologue of cxcl-1, is elevated in human IVD tissues surgically removed due to back pain.15 Cxcl-1 gene expression is elevated at 2 days and 2 wks after injury (n = 5, P = 0.023 and 0.010, respectively) (Fig. 4B). There is no statistically significant change at 1 wk or 4 wks after injury (n = 5, P = 0.850; n = 4, P = 0.879, respectively) (Fig. 4B).
Extracellular Matrix Gene Expression Representing Injury-Repair Responses To examine whether expression of genes encoding extracellular matrix molecules is altered in response to injury, we examined collagen1 α1 chain (col1a1) and collagen2 α1 chain (col2a1) gene expression. col1a1 gene expression was downregulated at day 2 after injury (n = 5, P = 0.023) (Fig. 4C) but upregulated at 2 wks after injury (n = 5, P = 0.010). There is no statistically significant difference in col1a1 gene expression at 1 wk or 4 wks after injury (n = 5, P = 0.850 or 0.879, respectively) (Fig. 4C). col2a1 gene expression comparing © 2017 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 2. Histological changes 2 days, 1, 2, and 4 wks after coccygeal IVD injury. Injured and adjacent intact IVDs were stained with Alcian blue and counterstained with H&E; A′–D′ and A″–D″ are magnified images in squares in A–D, respectively. E′–H′ and E″–H″ are magnified images in squares in E–H, respectively. Scale bars in panels A–H represent 200 μm; scale bars in panels A′–H′ and A″–H″ represent 50 μm.
injured and intact controls did not reach statistical significance at any of the time points studied (n = 4–5, P > 0.050) (Fig. 4D).
DISCUSSION The mouse model is attractive because of the opportunity for genetic manipulations. However, the mouse tail is nonweight bearing; thus, the biomechanics of the vertebra-disc-vertebra motion segment differ from those of the human lumbar spine. TABLE 3. Histological scores Scores/Time Point Injured Intact P
2 days
1 wk
2 wks
4 wks
2.56 (0.48) 3.11 (0.97) 3.56 (1.11) 5.78 (0.73) 0.56 (0.29) 0.56 (0.11) 0.33 (0.19) 0.44 (0.11) 0.1019 0.0987 0.0772 0.0241
Data are presented as mean (SEM).
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Nonhuman primates are the only mammalian species that spend part of their time in an upright position and share important physiological characteristics with humans. However, rodent models provide opportunities for genetic manipulation, and a larger sample number can be achieved. Thus, these are useful in spine research. The needle injury resulted in a full thickness AF injury model with a relatively large needle (26 G), which results in an acute NP herniation through the injured AF. Although this is an important model to study disc herniation and the subsequent degeneration, we acknowledge that only a percentage of the human condition involves concurrent NP herniation while others only manifest as annular bulge. Furthermore, variability of the procedure has been taken into consideration, even with meticulous training and practice on the part of the surgeon. The degeneration induced by puncture with a 26-G needle was mild to moderate, equivalent to grade 1 in the goat (grades 0 being normal, 2 being severe)17 and grade 2 to 4 in humans (grade 1 being normal, grade 5 being severe).24 Not www.ajpmr.com
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FIGURE 3. Aggrecan neoepitope 1 and 4 wks after coccygeal IVD injury. Injured and adjacent intact IVDs were stained with antibody that recognizes the aggrecan neoepitope VDIPEN and were counterstained with hematoxylin. The scale bar represents 625 μm in panels A–D, 125 μm in panels A′–D′.
surprisingly, injury induced in our model is more severe than that reported with a thinner needle.11 By 4 wks after injury, the NP space was filled with chondrocyte-like cells (Figs. 1, 2). It is worth noting that although histological features continue to be the standard criterion for evaluating human disc degeneration, the scores tend to be subjective and insensitive to detect subtle differences between injured groups. In addition, because the severity of injury in the mouse model can be controlled by using different sized needles, a smaller gauge needle may be used for experiments that aim to detect subtle changes. The origin of the cells filling up the space left by NP herniation has not been determined yet; they may be proliferating residual NP cells or migrating from the AF or surrounding cartilaginous endplate as previously suggested.25 One important mechanistic question is the origin of cells contributing to repair. Cell tracing method with a fluorescent Cre reporter mouse crossed with Cre specifically expressed in different zones of the mouse IVD (e.g., Col1CreER/Col2CreER/Shh-creER) would definitively confirm which anatomical area these cells migrated from. Col1CreER,26 Col2CreER27 or ShhCreER28 would allow specific fluorescent activities in the outer AF, inner AF, or NP, respectively. Adopting this methodology would help
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determine the cell origin and elucidate the mechanism of repair after a disc injury. Aggrecanase and multiple MMPs are capable of cleaving aggrecan.22,23 The neoepitope VDIPEN resulting from MMP cleavage21 is detected consistently after disc injury (Fig. 3). The findings suggest that MMPs, at least in part, play a role in tissue remodeling, degeneration, and repair after an IVD injury. Although the ultimate goal of the study is to understand the pathophysiological events in human IVD degeneration, we acknowledge that there are significant differences between mouse and human spine both histologically16 and physiologically. Notochordal cells persist in the mouse IVD, which play a significant role in development of IVD degeneration.29 Other significant differences in mechanical loading and composition and metabolism exist.1,30 The adult goat/sheep and chondrodystrophic dogs do not have persistent notochord cells and thus are more suitable for certain studies. However again, these animals lack the opportunity for genetic manipulations that the mouse model offers. Adam8 (also known as CD156) belongs to the Adam family of cell surface proteases.13 Members of the Adam family participate in remodeling of extracellular matrix, cell migration, and processing of membrane-bound signaling molecules31 and © 2017 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 4. Adam8, cxcl1, type 1 collagen (col1a1), and type 2 collagen (col2a1) gene expression after injury. A, Adam8 to gapdh gene expression ratio. B, cxcl1 to gapdh gene expression ratio. C, col1a1 to gapdh gene expression ratio. D, Type 2 collagen (col2a1) to gapdh gene expression ratio. Each individual symbol within a column represents injured and its adjacent intact IVD (control) from one mouse. *P < 0.05. **P < 0.01.
play important roles in the immune system. Adam8 is known to cleave fibronectin and promote cartilage degradation in articular cartilage.32 In human IVD tissue, adam8 has been shown to cleave fibronectin, resulting in a bioactive fragment14 that could further accelerate disc degeneration.33,34 Adam8 gene expression is elevated at all time points examined, suggesting a key role in the injury/repair process, thus warranting further investigation. However, we have yet to confirm the key role of adam8 in the repair process after IVD injury. For example, adam8 protein level and tissue distribution need to be determined by quantitative Western blot and immunostaining, to confirm that the messenger RNAs are indeed translated into protein. We have shown clear progression of IVD degeneration in response to tail IVD injury by histology, immunostaining, and gene expression methods. One limitation is that although we found that aggrecan neoepitope was present in areas rich in chondrocyte-like cells, cellularity has not been quantified. To examine cell proliferation, we will use a method detecting the incorporation of 5-ethynyl-2′-deoxyuridine, a thymidine analog, into DNA,35 as previously reported.36,37 In summary, we have established a minimally invasive approach to the mouse tail IVD injury model that is easier to use than the previously described open surgical approach. Adam8 gene expression is upregulated at all time points after injury, suggesting a role of this protease in initiating injury/ repair events. This IVD injury model may be suitable for elucidating the origin and nature of the cells appearing in injured NP and the role of adam8 in cell migration/inflammation and aggrecan degradation.
ACKNOWLEDGMENTS The authors thank Debra Pawlowski, Jeffrey House, and Pierre Conti, VMD, for care of the animals. The authors also © 2017 Wolters Kluwer Health, Inc. All rights reserved.
thank Jason W. Ashley, PhD, Haoru Jia, MD, and John T. Martin, PhD, for technical support, Martin F. Heyworth, MD, for critically editing the article, and Carla R. Scanzello for valuable insights and discussions.
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12. Yoshida S, Setoguchi M, Higuchi Y, et al: Molecular cloning of cDNA encoding MS2 antigen, a novel cell surface antigen strongly expressed in murine monocytic lineage. Int Immunol 1990;2:585–91 13. Yoshiyama K, Higuchi Y, Kataoka M, et al: CD156 (human ADAM8): expression, primary amino acid sequence, and gene location. Genomics 1997;41:56–62 14. Ruel N, Markova DZ, Adams SL, et al: Fibronectin fragments and the cleaving enzyme ADAM-8 in the degenerative human intervertebral disc. Spine (Phila Pa 1976) 2014;39:1274–9 15. Zhang Y, Chee A, Shi P, et al: Intervertebral disc cells produce interleukins found in patients with back pain. Am J Phys Med Rehabil 2016;95:407–15 16. Zhang Y, Lenart BA, Lee JK, et al: Histological features of endplates of the mammalian spine: from mice to men. Spine (Phila Pa 1976) 2014;39:E312–7 17. Zhang Y, Drapeau S, An HS, et al: Histological features of the degenerating intervertebral disc in a goat disc-injury model. Spine (Phila Pa 1976) 2011;36:1519–27 18. Ye J, Coulouris G, Zaretskaya I, et al: Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 2012;13:134 19. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 2001;25:402–8 20. Schmittgen TD, Livak KJ: Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008;3:1101–8 21. Flannery CR, Lark MW, Sandy JD: Identification of a stromelysin cleavage site within the interglobular domain of human aggrecan. Evidence for proteolysis at this site in vivo in human articular cartilage. J Biol Chem 1992;267:1008–14 22. Nagase H, Kashiwagi M: Aggrecanases and cartilage matrix degradation. Arthritis Res Ther 2003;5:94–103 23. Roughley PJ, Mort JS: The role of aggrecan in normal and osteoarthritic cartilage. J Exp Orthop 2014;1:8 24. Thompson JP, Pearce RH, Schechter MT, et al: Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc. Spine (Phila Pa 1976) 1990;15: 411–5 25. Kim KW, Lim TH, Kim JG, et al: The origin of chondrocytes in the nucleus pulposus and histologic findings associated with the transition of a notochordal nucleus pulposus to a
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fibrocartilaginous nucleus pulposus in intact rabbit intervertebral discs. Spine (Phila Pa 1976) 2003;28:982–90 Bedore J, Quesnel K, Quinonez D, et al: Targeting the annulus fibrosus of the intervertebral disc: Col1a2-cre(ER)T mice show specific activity of cre recombinase in the outer annulus fibrosus. J Cell Commun Signal 2016;10:137–42 Kondo N, Yuasa T, Shimono K, et al: Intervertebral disc development is regulated by Wnt/ β-catenin signaling. Spine (Phila Pa 1976) 2011;36:E513–8 Choi KS, Cohn MJ, Harfe BD: Identification of nucleus pulposus precursor cells and notochordal remnants in the mouse: implications for disk degeneration and chordoma formation. Dev Dyn 2008;237:3953–8 McCann MR, Séguin CA: Notochord cells in intervertebral disc development and degeneration. J Dev Biol 2016;4:1–18 Lotz JC: Animal models of intervertebral disc degeneration: lessons learned. Spine (Phila Pa 1976) 2004;29:2742–50 Hernandez I, Moreno JL, Zandueta C, et al: Novel alternatively spliced ADAM8 isoforms contribute to the aggressive bone metastatic phenotype of lung cancer. Oncogene 2010;29:3758–69 Zack MD, Arner EC, Anglin CP, et al: Identification of fibronectin neoepitopes present in human osteoarthritic cartilage. Arthritis Rheum 2006;54:2912–22 Anderson DG, Li X, Balian G: A fibronectin fragment alters the metabolism by rabbit intervertebral disc cells in vitro. Spine (Phila Pa 1976) 2005;30:1242–6 Anderson DG, Markova D, Adams SL, et al: Fibronectin splicing variants in human intervertebral disc and association with disc degeneration. Spine (Phila Pa 1976) 2010;35:1581–8 Buck SB, Bradford J, Gee KR, et al: Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2'-deoxyuridine antibodies. Biotechniques 2008;44:927–9 Candela ME, Cantley L, Yasuaha R, et al: Distribution of slow-cycling cells in epiphyseal cartilage and requirement of β-catenin signaling for their maintenance in growth plate. J Orthop Res 2014;32:661–8 Candela ME, Wang C, Gunawardena AT, et al: Alpha 5 integrin mediates osteoarthritic changes in mouse knee joints. PLoS One 2016;11:e0156783
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Intervertebral Disc Degeneration in a Percutaneous Mouse Tail Injury Model Zuozhen Tian, MS, Xiaoyuan Ma, MD, Miersalijiang Yasen, MD, PhD, Robert L. Mauck, PhD, Ling Qin, PhD, Frances S. Shofer, PhD, Lachlan J. Smith, PhD, Maurizio Pacifici, PhD, Motomi Enomoto-Iwamoto, DDS, PhD, and Yejia Zhang, MD, PhD Objectives: Intervertebral disc (IVD) degenerates progressively with age and after injuries. In this study, we aimed to characterize early molecular events underlying disc degeneration using a mouse tail IVD injury model. Design: We have established a transcutaneous minimally invasive approach to induce mouse tail IVD injury under fluoroscopic guidance. Morphological and molecular changes in the injured IVDs are compared with the baseline features of adjacent intact levels. Results: After needle puncture, tail IVDs exhibited time-dependent histological changes. The aggrecan neoepitope VDIPEN was evident from 2 days to 4 wks after injury. A disintegrin and metalloproteinase domain-containing protein 8 (adam8) is a surface protease known to cleave fibronectin in the IVD. Gene expression of adam8 was elevated at all time points after injury, whereas the increase of C-X-C motif chemokine ligand (cxcl)-1 gene expression was statistically significant at 2 days and 2 wks after injury. Type 1 collagen gene expression decreased initially at day 2 but increased at 2 wks after injury, whereas no significant change in type 2 collagen gene expression was observed. The extracellular matrix gene expression pattern is consistent with fibrocartilage formation after injury. Conclusions: Mouse tail IVDs degenerate after needle puncture, as demonstrated by histological changes and aggrecan degradation. The minimally invasive tail IVD injury model should prove useful to investigators studying mechanisms of IVD degeneration and repair. Key Words: Intervertebral Disc, Mouse Model, Injury, Degeneration, A Disintegrin and Metalloproteinase Domain-Containing Protein 8 (Am J Phys Med Rehabil 2017;00:00–00)
n humans, because most patients do not receive medical attention for back pain during the early stages of intervertebral disc (IVD) degeneration, little is known about the changes that occur during the initial phases of such degeneration. Animal models of disc degeneration would be useful in understanding the events that initiate inflammation/repair and in identifying critical steps that may be amenable to intervention.1 This work aims to define the early molecular changes during the initial 4 wks, concurrent with histological changes after an injury to the tail IVD in the mouse. The IVD injury model has been widely used to study the course of IVD degeneration, in large animals (ovine,2 porcine3),
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dogs,4 and small animals (rabbit,5–8 rat,9 and mouse10,11). One main advantage of the mouse model is the ability to perform genetic manipulations during IVD degeneration. Challenges of working with the mouse IVD mainly reflect its small size: surgical precision is crucial, and the amount of tissue for molecular and biochemical assays is limited. The IVD histology and molecular changes after injury with a 29-gauge (G) needle were first described by Yang et al.11 The deterioration of biomechanical properties along with histological changes after tail IVD injury with a 26-G needle were further described by Martin et al.10 The needle puncture, with a 26-G needle, was severe enough to induce IVD degeneration detectable with biomechanical
From the Department of Physical Medicine & Rehabilitation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (ZT, MY, RLM, YZ); Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (XM, RLM, LQ, LJS, YZ); Translational Musculoskeletal Research Center, Corporal Michael J. Crescenz Veterans Affairs Medical Center, Philadelphia, Pennsylvania (RLM, LJS, YZ); Department of Emergency Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (FSS); Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (LJS); Department of Surgery, Division of Orthopaedic Surgery, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania (MP, ME-I); and Department of Orthopaedics, University of Maryland, Baltimore, Maryland (ME-I). All correspondence and requests for reprints should be addressed to: Yejia Zhang, MD, PhD, Department of Physical Medicine & Rehabilitation, Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, and Translational Musculoskeletal Research Center, Corporal Michael J. Crescenz Veterans Affairs Medical Center, Philadelphia, PA 19104. YZ has been supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (1K08 HD049598). This work is supported, in part, by research grants from the North American Spine Society, a pilot grant
from the Department of Veterans Affairs Healthcare Network-VISN 4, and a Penn Center for Musculoskeletal Disorders pilot grant (P30-AR050950-10 Pilot). All authors critically reviewed and approved the article. The Thomas Jefferson University Sidney Kimmel Cancer Center Translational Research/Pathology Shared Resource Core Facility provided outstanding histology service. None of the authors have any professional and financial affiliation that could be perceived to be biased in the presentation of the current work. ZT, MY, XM, LQ, ME-I, and YZ contributed in data acquisition. ZT, YZ, MP, ME-I, LJS, and RLM contributed in research design and/or data interpretation. FSS contributed in statistical analysis and research design. YZ, MP, ME-I, LJS, RLM, and LQ drafted and revised the article. YZ and ZT are responsible for storage and access of all primary data. All authors have read and approved the final submitted article. Financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.ajpmr.com). Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0894-9115 DOI: 10.1097/PHM.0000000000000818
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measures.10 The advantages of the tail injury method include easy access to the tail disc, low morbidity, and a reproducible course of degeneration.
Primary Objective The main objective of this study was to further refine the surgical technique, including percutaneous puncture of the disc, thus shortening and simplifying the procedure and reducing mouse suffering. As a result of reduced surgical trauma, confounding factors are also reduced in the examination of inflammatory markers. Using this IVD injury model, we determined the time course of histopathological changes and changes in gene expression of adam8, which is an important molecule in inflammation12,13 and is upregulated in degenerated IVDs in humans.14 We also examined cxcl-1 gene expression after injury because we have previously shown that interleukin 8, the human homologue of cxcl-1, is elevated in human IVD tissues surgically removed because of back pain.15
MATERIALS AND METHODS Mice All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Corporal Michael J. Crescenz Veterans Affairs Medical Center in Philadelphia (approval # 01517-0002). Fifteen young adult (8 wk old) female C57BL/6j mice (the Jackson Laboratory, Bar Harbor, ME) were used in this study. Mice were housed under pathogen-free conditions with environmental enrichment, with up to five animals per cage. Twenty animals were used for RNA extraction and histology, with five animals at each of four time points.
Tail Injury Surgery The mouse was anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg) subcutaneously. Under anesthesia, the mouse tail IVDs were injured with a 26-G needle inserted under fluoroscopic guidance with a mini C-arm (OrthoScan FD Pulse Mini C-Arm, Orthoscan Inc, Scottsdale, AZ). Specifically, the skin was cleaned with betadine, the mouse coccygeal
(Co) IVDs were identified, and a 26-G needle was inserted into the IVD space until the needle tip reached 2/3 of the disc thickness (Fig. S1, Supplemental Digital Content 1, http://links. lww.com/PHM/A472). Gelatinous tissues were often found on the needle tip after this had been removed, suggesting that the needle puncture induces an acute herniation of the gelatinous nucleus pulposus (NP) of the IVD. An arrow indicates the coccygeal disc between the third and fourth coccygeal vertebrae (Co3/4; arrows in Fig. S1, Supplemental Digital Content 1, http://links.lww.com/PHM/A472). This procedure has been modified from previously published work,10 where a skin incision was made in the tail over the IVD before needle insertion into the disc. In the current study, the Co3/4 and Co5/6 IVDs in each mouse were injured, whereas Co4/5 and Co6/7 served as intact controls (Fig. S1, Supplemental Digital Content 1, http://links.lww.com/PHM/A472). The percutaneous approach reduced procedure time and minimized risk to the animals. The animals behaved normally (by observing breathing, eating, ambulation, etc.) and did not require medication for pain on the day after surgery. No adverse event such as infection or bleeding was noted. Five animals were sacrificed by exposure to CO2 at each of the following time points: 2 days, 1 wk, 2 wks, and 4 wks after tail disc injury. From each mouse tail, Co3/4 (injured) and Co4/5 (intact control) discs were isolated individually for RNA extraction. Co5/6 (injured) and Co6/7 (intact control) were isolated en bloc (Fig. 1) for histological examination and immunostaining (Fig. 3).
Histological Evaluation The IVDs and portions of the adjacent bony vertebral bodies were isolated immediately after euthanasia. The disc with its surrounding vertebral bodies was fixed with 4% paraformaldehyde for 24 hrs. The bone-disc-bone segments were decalcified with a solution consisting of 10% citric acid with 22% formic acid for 24 hrs until the bony portion was completely decalcified, as previously described.16 The tissues were then dehydrated and embedded in paraffin and sectioned to 5-μM thickness, and the sections were stained with Alcian blue with hematoxylin and eosin (H&E) counter stain or with H&E only. Specifically, the tissue sections were stained with
FIGURE 1. Histological changes (low magnification) 2 days, 1, 2, and 4 wks after coccygeal IVD injury. Injured (outlined in red) and the adjacent intact (outlined in green) IVDs were stained with Alcian blue and counterstained with H&E. Scale bars represent 1 mm.
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1% Alcian blue solution (Poly Scientific R&D Corp., Bay Shore, NY) for 5 minutes, followed by hematoxylin for 5 mins and eosin for approximately 20 secs. All samples were examined under a light microscope (Nikon) and photographed. Representative sections from each time point were chosen for presentation.
Histological Grades A modified histological grading scale for mouse discs was developed based on the previous grading methods.11,17 As shown in Table 1, a numeric scale for quantification of the degree of degeneration was designed. Analysis included the status of the annulus fibrosus (AF) organization and the extracellular matrix and cell morphology in the NP. Total scores ranged from 0 to 8. Scores of 0 to 2.0 represent essentially normal tissue; IVDs with mild disc degeneration scored 2.1 to 4.0 points; IVDs with moderate degeneration scored 4.1 to 6.0; IVDs with severe degeneration scored 6.1 to 8.0. Sections of injured and intact discs from 12 mice were evaluated by three experienced clinician scientists in a double-blinded manner. The total scores for NP and AF were calculated, and interobserver variability was assessed by a weighted Kappa using SAS software (Version 9.4; SAS Institute, Cary, NC). Average scores of all three raters were used to compare injured and intact disc histological grades.
Immunohistochemical Staining Aggrecan neoepitope was examined with antibody to its cleavage site, with the VDIPEN neoepitope at the cleavage site. Specifically, the paraffin sections were pretreated with 0.1% pepsin with 0.02 N HCl and incubated with primary antibodies (a generous gift from Dr. J. Mort, Shriner's Hospital) at 1:1000 dilution. The antibodies were visualized by incubation with appropriate biotinylated antibodies (Vector Laboratories, Burlingame, CA) followed by the color development method using the ImmpPCAT DAB Peroxidase Substrate kit (Vector Laboratories, Burlingame, CA). The sections were counterstained with hematoxylin.
RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction Intervertebral disc tissues were separated from their adjacent cartilaginous endplates and bone with a scalpel, under a
TABLE 1. Histological scoring criteria Grade 0 1 2 3 4
AF
NP
Discrete and well-apposed lamellae Moderately serpentine with rupture Severely serpentine with rupture, reversed contour Indistinct border of NP and AF Clefts formation; lamella fragmentation
Oval shape, highly hydrated, many notochordal cells Irregular shape, decreased hydration Large defects, cell clusters present Minor clefts, cell clusters decrease Occupied by connective tissue
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dissecting microscope (VistaVision; VWR International, Radnor, PA) (Fig. S2, Supplemental Digital Content 2, http://links.lww. com/PHM/A473). Total cellular RNA was isolated by the Trizol method as described previously.8 Specifically, the isolated IVD tissues were soaked in RNALater (Ambion, Foster City, CA) overnight, and stored at −80°C until extraction. On the day of RNA extraction, RNALater was removed and the tissues were snap frozen with liquid nitrogen, then transferred into Trizol (Invitrogen, Carlsbad, CA). The tissues were homogenized with a homogenizer, with disposable OmniTip probes for hard tissue (Omin International, Kennesaw, GA). Phenol was then extracted with chloroform three times and discarded. RNA was precipitated with 70% ethanol and was further purified using an RNeasy Micro Kit (Qiagen), according to the manufacturer's protocol. RNA concentration was determined using a Synergy H4 Hybrid Reader (BioTek, Winooski, VT). To generate cDNA, all RNA from each IVD (7–20 ng/μl, total volume 50 μl per sample) was used as template in a reverse transcriptase reaction using the SuperScript VILO cDNA synthesis kit (Life Technologies, Carlsbad, CA) containing random hexamers and added polyDT primers (Invitrogen, Carlsbad, CA). CDNA sequences were retrieved from Ensembl (release 84, March 2016). Primers for real-time polymerase chain reaction (PCR) (Table 2) were designed using Primer-BLAST18 and synthesized by Invitrogen (Carlsbad, CA) (Table 2). For each PCR reaction, cDNA, SYBR Select master mix (Life Technologies, Carlsbad, CA), and primers (working concentration 0.5 μM) were mixed, and deionized water was added to a total volume of 20 μl per reaction. MicroAmp Optical 96-well reaction plates (Applied Biosystems, Foster City, CA) with 20 μl of reaction mix/well were sealed with optical adhesive film (Life Technologies, Frederick, MD) and run in a ViiA7 real-time PCR system (Applied Biosystems, Foster City, CA) using the following program: (1) 50°C for 2 mins, (2) 95°C for 2 mins, (3) 95°C for 15 secs, (4) 58°C for 1 min, (5) repeat steps 3 and 4 for a total of 40 times. Single products were confirmed by determining melting curves at the conclusion of the reaction. Relative expression was calculated using the 2−ΔΔCt method19,20 normalized to gapdh (endogenous loading control).
Statistics The difference in genes of interest (adam8, cxcl1, col1a1, and col2a1) and house-keeping gene (gapdh) cycle thresholds (ΔCT) was calculated for each injured/intact pair. To assess differences in ΔCT between injured and intact tissues for each time period, a two-factor analysis of variance in repeated measures was used, where time was a grouping factor and injured/ intact was the repeated measure. Post hoc t tests using the pooled variance were performed for injured/intact differences within each time period. A P value of less than 0.05 was considered statistically significant. All analyses were performed using SAS statistical software (Version 9.4; SAS Institute, Cary, NC).
RESULTS Histology of Injured Mouse Tail IVD After injury, the IVD tissue showed loss of normal NP architecture (Figs. 1, 2). During the initial postinjury period www.ajpmr.com
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TABLE 2. Primers for real-time PCR Gene
Sequence
Expected Product
Transcript ID
Gapdh
F: 5′-AAG GGC TCA TGA CCA CAG TC-3′ R: 5′-GGA TGA CCT TGC CCA CAG-3′ F: 5′-TTG GCA CAG GCA GCA ACA TT-3′ R: 5′-CAC TCC CTC TTG TGG TTG CA-3′ F: 5′-CTG ACG CAT GGC CAA GAA GA-3′ R: 5′-ATA CCT CGG GTT TCC ACG TC-3′ F: 5′-CTG GGA ATG TCC TCT GCG ATG-3′ R: 5′-GCC CCT TTG GCC CTA ATT TTC-3′ F: 5′-TGCACCCAAACCGAAGTCAT-3′ R: 5′-TTGTCAGAAGCCAGCGTTCAC-3′
133 bp
ENSMUST00000118875 NM_008084 ENSMUST00000106069 NM_007403 ENSMUST00000001547 NM_007742 ENSMUST00000023123 NM_031163 ENSMUST00000031327 NM_008176
Adam8 Col1a1 Col2a1 Cxcl1
168 bp 72 bp 135 bp 177 bp
ENSMUST, Ensembl ID; F, forward; NM, The National Center for Biotechnology Information reference sequence; R, reverse.
(2 days to 2 wks), the empty spaces in the NP are most likely due to herniated NP, although this could also be the result of sectioning artifact (Fig. 2, red squares in 2-day, 1-wk and 2-wk panels and Figs. 2A–C and A′–C′, arrows). The whole IVD changed from an oval shape (Figs. 2E–H) to an irregular shape (Figs. 2A–D), suggesting loss of NP turgor, as a result of decompression. By 4 wks after injury, the IVD space was filled with chondrocyte-like cells (Figs. 2D, D′) positive to Alcian blue staining. The AF structure also changed from well-organized lamellae (Figs. 2E–H, yellow squares E″–H″) to severely serpentine layers with clefts (Figs. 2A–D, yellow squares and A″–D″). We evaluated histologic grade of the injured and control IVDs, modified from the grading system reported previously (Table 1).11 The scoring is based on microscopic sections, stained with Alcian blue and H&E (Fig. 2), and with H&E only (images not shown). Nucleus pulposus or AF showed grades ranging from 0 (normal) to 4 (severely degenerative), with the total score (NP + AF scores) ranging from 0 to 8 (Table 1). The scores of 3 graders correlated well, as measured by interrater variability (κ = 0.9311). The average scores for the intact IVDs ranged from 0.33 to 0.56, whereas those for the injured IVDs increased from 2.56 to 5.78 over time after the surgery (Table 3). The scores for intact discs were all in the normal range (≤2.0), whereas the scores for injured discs were in the mild (2.1–4.0) or moderate (4.1–6.0) range of degeneration according to the grading system described (Table 1). Differences between the histological scores of injured IVDs at 2 days, 1 wk, and 2 wks after injury, compared with those of intact IVDs, were not statistically significant (n = 3, P = 0.102, 0.099, and 0.077, respectively) (Table 3). The difference in histological score between injured and intact control IVDs was statistically significant at 4 wks after injury (n = 3, P = 0.0241) (Table 3).
Increase in Cleaved Products of Aggrecan Associated With Cartilaginous Changes Immunohistochemical examination was performed with an antibody that recognizes the aggrecan neoepitope, likely generated by matrix metalloproteinases (MMPs).21–23 Immunoreactivity was found in the regions rich in chondrocyte-like
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cells in injured IVD at 1 and 4 wks after injury (Figs. 3A, B, A′, B′; blue arrows), whereas the staining was limited to the interface between the NP and AF in intact IVDs (Fig. 3, intact′; blue arrows), suggesting that proteoglycan remodeling becomes active in the cartilaginous lesion in injured IVD.
Adam8 Gene Expression Is Upregulated After Injury We have previously demonstrated that disc degeneration is associated with increased adam8 protease and its fibronectin cleavage products in humans.14 To determine whether this phenomenon is recapitulated in this IVD injury model, adam8 expression was measured via quantitative real-time PCR for a 4-wk period after injury (Fig. 4A). After injury, adam8 gene expression increased compared with adjacent intact controls at all time points examined: day 2, P = 0.004; 1 wk, P = 0.018; 2 wks, P = 0.003; and 4 wks, P = 0.025 (n = 5 at each time point) (Fig. 4A).
Cxcl-1 Gene Expression Is Upregulated After Injury We have previously shown that interleukin 8, the human homologue of cxcl-1, is elevated in human IVD tissues surgically removed due to back pain.15 Cxcl-1 gene expression is elevated at 2 days and 2 wks after injury (n = 5, P = 0.023 and 0.010, respectively) (Fig. 4B). There is no statistically significant change at 1 wk or 4 wks after injury (n = 5, P = 0.850; n = 4, P = 0.879, respectively) (Fig. 4B).
Extracellular Matrix Gene Expression Representing Injury-Repair Responses To examine whether expression of genes encoding extracellular matrix molecules is altered in response to injury, we examined collagen1 α1 chain (col1a1) and collagen2 α1 chain (col2a1) gene expression. col1a1 gene expression was downregulated at day 2 after injury (n = 5, P = 0.023) (Fig. 4C) but upregulated at 2 wks after injury (n = 5, P = 0.010). There is no statistically significant difference in col1a1 gene expression at 1 wk or 4 wks after injury (n = 5, P = 0.850 or 0.879, respectively) (Fig. 4C). col2a1 gene expression comparing © 2017 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 2. Histological changes 2 days, 1, 2, and 4 wks after coccygeal IVD injury. Injured and adjacent intact IVDs were stained with Alcian blue and counterstained with H&E; A′–D′ and A″–D″ are magnified images in squares in A–D, respectively. E′–H′ and E″–H″ are magnified images in squares in E–H, respectively. Scale bars in panels A–H represent 200 μm; scale bars in panels A′–H′ and A″–H″ represent 50 μm.
injured and intact controls did not reach statistical significance at any of the time points studied (n = 4–5, P > 0.050) (Fig. 4D).
DISCUSSION The mouse model is attractive because of the opportunity for genetic manipulations. However, the mouse tail is nonweight bearing; thus, the biomechanics of the vertebra-disc-vertebra motion segment differ from those of the human lumbar spine. TABLE 3. Histological scores Scores/Time Point Injured Intact P
2 days
1 wk
2 wks
4 wks
2.56 (0.48) 3.11 (0.97) 3.56 (1.11) 5.78 (0.73) 0.56 (0.29) 0.56 (0.11) 0.33 (0.19) 0.44 (0.11) 0.1019 0.0987 0.0772 0.0241
Data are presented as mean (SEM).
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Nonhuman primates are the only mammalian species that spend part of their time in an upright position and share important physiological characteristics with humans. However, rodent models provide opportunities for genetic manipulation, and a larger sample number can be achieved. Thus, these are useful in spine research. The needle injury resulted in a full thickness AF injury model with a relatively large needle (26 G), which results in an acute NP herniation through the injured AF. Although this is an important model to study disc herniation and the subsequent degeneration, we acknowledge that only a percentage of the human condition involves concurrent NP herniation while others only manifest as annular bulge. Furthermore, variability of the procedure has been taken into consideration, even with meticulous training and practice on the part of the surgeon. The degeneration induced by puncture with a 26-G needle was mild to moderate, equivalent to grade 1 in the goat (grades 0 being normal, 2 being severe)17 and grade 2 to 4 in humans (grade 1 being normal, grade 5 being severe).24 Not www.ajpmr.com
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FIGURE 3. Aggrecan neoepitope 1 and 4 wks after coccygeal IVD injury. Injured and adjacent intact IVDs were stained with antibody that recognizes the aggrecan neoepitope VDIPEN and were counterstained with hematoxylin. The scale bar represents 625 μm in panels A–D, 125 μm in panels A′–D′.
surprisingly, injury induced in our model is more severe than that reported with a thinner needle.11 By 4 wks after injury, the NP space was filled with chondrocyte-like cells (Figs. 1, 2). It is worth noting that although histological features continue to be the standard criterion for evaluating human disc degeneration, the scores tend to be subjective and insensitive to detect subtle differences between injured groups. In addition, because the severity of injury in the mouse model can be controlled by using different sized needles, a smaller gauge needle may be used for experiments that aim to detect subtle changes. The origin of the cells filling up the space left by NP herniation has not been determined yet; they may be proliferating residual NP cells or migrating from the AF or surrounding cartilaginous endplate as previously suggested.25 One important mechanistic question is the origin of cells contributing to repair. Cell tracing method with a fluorescent Cre reporter mouse crossed with Cre specifically expressed in different zones of the mouse IVD (e.g., Col1CreER/Col2CreER/Shh-creER) would definitively confirm which anatomical area these cells migrated from. Col1CreER,26 Col2CreER27 or ShhCreER28 would allow specific fluorescent activities in the outer AF, inner AF, or NP, respectively. Adopting this methodology would help
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determine the cell origin and elucidate the mechanism of repair after a disc injury. Aggrecanase and multiple MMPs are capable of cleaving aggrecan.22,23 The neoepitope VDIPEN resulting from MMP cleavage21 is detected consistently after disc injury (Fig. 3). The findings suggest that MMPs, at least in part, play a role in tissue remodeling, degeneration, and repair after an IVD injury. Although the ultimate goal of the study is to understand the pathophysiological events in human IVD degeneration, we acknowledge that there are significant differences between mouse and human spine both histologically16 and physiologically. Notochordal cells persist in the mouse IVD, which play a significant role in development of IVD degeneration.29 Other significant differences in mechanical loading and composition and metabolism exist.1,30 The adult goat/sheep and chondrodystrophic dogs do not have persistent notochord cells and thus are more suitable for certain studies. However again, these animals lack the opportunity for genetic manipulations that the mouse model offers. Adam8 (also known as CD156) belongs to the Adam family of cell surface proteases.13 Members of the Adam family participate in remodeling of extracellular matrix, cell migration, and processing of membrane-bound signaling molecules31 and © 2017 Wolters Kluwer Health, Inc. All rights reserved.
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Percutaneous Mouse Tail Injury Model
FIGURE 4. Adam8, cxcl1, type 1 collagen (col1a1), and type 2 collagen (col2a1) gene expression after injury. A, Adam8 to gapdh gene expression ratio. B, cxcl1 to gapdh gene expression ratio. C, col1a1 to gapdh gene expression ratio. D, Type 2 collagen (col2a1) to gapdh gene expression ratio. Each individual symbol within a column represents injured and its adjacent intact IVD (control) from one mouse. *P < 0.05. **P < 0.01.
play important roles in the immune system. Adam8 is known to cleave fibronectin and promote cartilage degradation in articular cartilage.32 In human IVD tissue, adam8 has been shown to cleave fibronectin, resulting in a bioactive fragment14 that could further accelerate disc degeneration.33,34 Adam8 gene expression is elevated at all time points examined, suggesting a key role in the injury/repair process, thus warranting further investigation. However, we have yet to confirm the key role of adam8 in the repair process after IVD injury. For example, adam8 protein level and tissue distribution need to be determined by quantitative Western blot and immunostaining, to confirm that the messenger RNAs are indeed translated into protein. We have shown clear progression of IVD degeneration in response to tail IVD injury by histology, immunostaining, and gene expression methods. One limitation is that although we found that aggrecan neoepitope was present in areas rich in chondrocyte-like cells, cellularity has not been quantified. To examine cell proliferation, we will use a method detecting the incorporation of 5-ethynyl-2′-deoxyuridine, a thymidine analog, into DNA,35 as previously reported.36,37 In summary, we have established a minimally invasive approach to the mouse tail IVD injury model that is easier to use than the previously described open surgical approach. Adam8 gene expression is upregulated at all time points after injury, suggesting a role of this protease in initiating injury/ repair events. This IVD injury model may be suitable for elucidating the origin and nature of the cells appearing in injured NP and the role of adam8 in cell migration/inflammation and aggrecan degradation.
ACKNOWLEDGMENTS The authors thank Debra Pawlowski, Jeffrey House, and Pierre Conti, VMD, for care of the animals. The authors also © 2017 Wolters Kluwer Health, Inc. All rights reserved.
thank Jason W. Ashley, PhD, Haoru Jia, MD, and John T. Martin, PhD, for technical support, Martin F. Heyworth, MD, for critically editing the article, and Carla R. Scanzello for valuable insights and discussions.
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Copyright © 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
